Factor X activation by the extrinsic pathway is pivotal for the initiation of blood coagulation. The enzyme complex (extrinsic Xase) that catalyses this reaction is composed of the serine proteinase, factor VIIa, and the integral membrane protein, tissue factor, that associate tightly on a cellular or phospholipid surface in a Ca2+-dependent reaction. We propose that the predominant contribution of interactions at extended macromolecular recognition sites (exosites) to the productive recognition of factors X and Xa by the Xase complex plays a central role in the regulation of enzymic function. These ideas establish a formal and experimental paradigm for the further investigation of Xase biochemistry in areas that remain poorly understood. We propose that the mechanism underlying the function of Xa-"activated" inhibitors of Xase, such as tissue factor pathway inhibitor (TFPI), derives from the ability of the inhibitor to bind Xa, which in turn can interact with Xase, to affect inhibitor delivery to the enzyme complex. Potent, irreversible inhibition results from the combined energetic contributions of exosite docking to the complex and the inhibition of the active site of Vila. We will develop a robust thermodynamic model to account for the contributions of the component reactions to the overall inhibition process. Key concepts will be tested by the use of recombinant derivatives of TFPI. In aim 2, we will pursue the hypotheses that distinct surfaces in the proteinase domain of factors XIXa participate in the exosite-dependent tethering interaction with Xase relative to those involved in the enzymic function of the product. We will test the idea that inhibitors, such as TFPI, exploit a prothrombin-like strategy to interact with Xa to yield an inhibitory scaffold that retains the ability to bind to Xase. In aim 3, we propose to utilize the established kinetic mechanism of factor X activation as a formal basis to investigate the function of tissue factor in factor X activation. We hypothesize that tissue factor-dependent modulation of binding interactions with factor X play a primary rather than ancillary role in the enhancing the perceived rate constant for catalysis by VITa. Finally, studies are proposed to examine the reaction pathway for the cleavage of the two bonds in factor IX by the Xase complex. We will assess the possibility that the Xase complex utilizes a common strategy of protein substrate recognition to accomplish the cleavage of both factors IX and X. These approaches will provide new insights into Xase regulation, with implications for enzymic function in normal hemostasis and in vascular disease.